research papers

192 http://dx.doi.org/10.1107/S2059798315024328 Acta Cryst. (2016). D72, 192–202

Received 5 October 2015

Accepted 17 December 2015

Edited by R. McKenna, University of

Florida, USA

Keywords: MERS-CoV; nucleocapsid; structure;

SAXS; RNA-binding domain.

PDB reference: N-terminal domain of the

MERS-CoV nucleocapsid, 4ud1

Supporting information: this article has

supporting information at journals.iucr.org/d

Structural characterization of the N-terminal partof the MERS-CoV nucleocapsid by X-ray diffractionand small-angle X-ray scattering

Nicolas Papageorgiou,a,b* Julie Lichiere,a,b Amal Baklouti,a,b Francois Ferron,a,b

Marion Sevajol,a,b Bruno Canarda,b and Bruno Coutarda,b*

aCNRS, AFMB UMR 7257, 13288 Marseille, France, and bAix-Marseille Universite, AFMB UMR 7257, 13288 Marseille,

France. *Correspondence e-mail: nicolas.papageorgiou@afmb.univ-mrs.fr, bruno.coutard@afmb.univ-mrs.fr

The N protein of coronaviruses is a multifunctional protein that is organized into

several domains. The N-terminal part is composed of an intrinsically disordered

region (IDR) followed by a structured domain called the N-terminal domain

(NTD). In this study, the structure determination of the N-terminal region of the

MERS-CoV N protein via X-ray diffraction measurements is reported at a

resolution of 2.4 A. Since the first 30 amino acids were not resolved by X-ray

diffraction, the structural study was completed by a SAXS experiment to

propose a structural model including the IDR. This model presents the

N-terminal region of the MERS-CoV as a monomer that displays structural

features in common with other coronavirus NTDs.

1. Introduction

Middle East respiratory syndrome coronavirus (MERS-CoV)

is the aetiological agent responsible for an outbreak of a

severe respiratory syndrome disease in the Middle East with

sporadic spread to Europe, Africa, Asia and America. Since

June 2012, the date of the first clinical case observed in Saudi

Arabia, more than 1600 other cases have been confirmed, with

a fatality rate of 36%, as reported by the World Health

Organization in December 2015. In humans, MERS-CoV can

result in asymptomatic viraemia to severe respiratory distress

associated with organ failure. The animal source of the virus is

still under investigation, but to date scenarios involving bats

and/or camels have been proposed (Azhar et al., 2014; Corman

et al., 2014; Muller et al., 2015; Raj et al., 2014).

MERS-CoV is a positive-strand RNA virus belonging to the

Betacoronavirus genus within the Coronaviridae family of

the Nidovirales order. The 50 two-thirds of the MERS-CoV

genome contains open reading frames 1a and 1b, which are

translated into the viral polyproteins pp1a and pp1ab,

respectively. The processing of these two proteins leads to 16

nonstructural proteins which are involved in the replication

complex. The 30-proximal third encodes the viral structural

proteins and several accessory proteins, which are translated

from a set of subgenomic mRNAs. Among the structural

proteins, the nucleocapsid (N) protein is a key protein

displaying several functions. Indeed, it is not only involved in

genome packaging through the formation of a ribonucleo-

protein complex (RNP; reviewed in Chang et al., 2014), but it

also regulates viral RNA synthesis during replication and

transcription (Chang et al., 2014; Nelson et al., 2000; Stohlman

et al., 1988; Wu et al., 2014). Furthermore, the N protein

ISSN 2059-7983

# 2016 International Union of Crystallography

participates in deregulation of the cell cycle, host translational

shutoff and innate immunity disruption (reviewed in McBride

et al., 2014).

The coronavirus N protein is a >40 kDa protein organized

into two main folded domains called the N-terminal domain

(NTD) and the C-terminal domain (CTD) separated by a

flexible linker (Fig. 1a). The NTD is subdivided into an RNA-

binding module preceded by an intrinsically disordered region

(IDR). This IDR contributes to the RNA-binding properties

of the NTD (Chang et al., 2009). The linker region contains a

Ser/Arg stretch that could correspond to phosphorylation sites

involved in the regulation of the functions of the N protein

(Chang et al., 2009; He et al., 2004; Peng et al., 2008; Surjit et al.,

2005; Wu et al., 2009). The CTD is involved in N-protein

dimerization and RNA binding (Chen et al., 2007; Takeda et

al., 2008; Yu et al., 2006). It is followed by another IDR. Based

on electron microscopy or small-angle X-ray scattering data,

the proposed structural models of the N-protein organization

suggest that the NTD and CTD may be independent domains

with no interaction between each other (Chang et al., 2006).

Since the full-length N protein contains a large fraction

(>35%) of disordered regions that are recalcitrant to crystal-

lization, it is therefore relevant to study the structural and

functional features of each domain independently.

The three-dimensional structures of the NTDs from several

coronaviruses have previously been determined: those from

Severe acute respiratory syndrome coronavirus (SARS-CoV;

Huang et al., 2004; Saikatendu et al., 2007), infectious bron-

chitis virus Beaudette (IBV; Fan et al., 2005; Jayaram et al.,

2006), murine hepatitis virus (MHV; Grossoehme et al., 2009)

and human coronavirus OC43 (HCoV-OC43; Chen et al.,

2013). An alignment between the NTD sequences of MERS-

CoV, IBVand SARS-CoV shows 37% sequence identity, while

all reported coronavirus NTD structures are organized as a

five-stranded antiparallel �-sheet

core domain with disordered

loops of varying sizes connecting

the strands. A positively charged

�-hairpin protrudes out of the

core domain. Structural analysis

enabled this region to be

proposed as a possible RNA-

binding domain. Indeed, muta-

tional analysis and NMR studies

led to the conclusion that this

region is a major actor in RNA

recruitment (Chen et al., 2013;

Clarkson et al., 2009; Grossoehme

et al., 2009; Saikatendu et al.,

2007; Tan et al., 2006). Recently,

an inhibitor of the RNA–N

interaction has been demon-

strated to have an antiviral effect

on HCoV-OC43 (Lin et al., 2014).

The coronavirus N protein is thus

an attractive target for antiviral

research. Because of the world-

wide spread of MERS-CoV, with

potential risks for healthcare, it is

important to accumulate struc-

tural and functional information

on MERS-CoV proteins in order

to provide tools for antiviral

development.

In this study, we present the

three-dimensional structure of

the MERS-CoV NTD with its

upstream IDR region (hereafter

referred to as NTD+) at a reso-

lution of 2.4 A obtained by X-ray

diffraction measurements as well

as a small-angle X-ray scattering

(SAXS) study of the protein in

solution in order to assess the

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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 193

Figure 1Sequence analysis of MERS-CoV N protein. (a) Modular organization of the coronavirus N protein. Thedomain boundaries correspond to the MERS-CoV N protein numbering. IDR, intrinsically disorderedregion; NTD, N-terminal domain; CTD, C-terminal domain. (b) Sequence alignment of the N-terminalregion of several N proteins for which the structure of the NTD has been documented. The sequences arenamed as follows: virus acronym_PDB code. The depicted secondary structure was retrieved from theMERS-CoV NTD+ structure described in this study. The alignment was obtained using T-Coffee(Notredame et al., 2000) and ESPript 3.0 (Robert & Gouet, 2014).

oligomerization state of MERS-CoV NTD+, and propose a

structural model of NTD+ including its disordered region.

2. Materials and methods

2.1. Protein production, purification and characterization

The codon-optimized DNA encoding the N-terminal region

(amino acids 1–164; NTD+) of the MERS-CoV nucleocapsid

(strain Betacoronavirus England 1, accession No. KC164505)

was synthesized by GenScript. The DNA sequence was

amplified by a two-step PCR before cloning into the expres-

sion vector pMCOX20A to enable the fusion of a cleavable

thioredoxin/6�His tag at the N-terminus of NTD+ (Lantez et

al., 2011). The protein was then produced in Escherichia coli

T7 Express (DE3) cells (New England Biolabs). Expression

was induced overnight at 290 K in TB medium with 0.5 mM

IPTG when the OD600 nm of the culture had reached 0.6.

Purification of the protein and tag removal was performed in

non-denaturing conditions as described previously (Lantez et

al., 2011). The final size-exclusion chromatography (SEC) step

was performed in 10 mM HEPES, 300 mM NaCl pH 7.5. The

protein folding and stability were assessed by thermal shift

assays (Geerlof et al., 2006) following a previously described

protocol (Malet et al., 2009). The nonspecific RNA-binding

properties of the MERS-CoV NTD+ were assessed by fluor-

escence polarization-based assays using 50-CCAGGCGACA-

UCAGCG-30 RNA labelled at the 30 end with a Cy3 probe.

The binding assay was performed in 50 mM Tris buffer pH 7.5,

1 mM DTT, 2 mM MgCl2. The Cy3-labelled RNA was used

at 50 nM. Fluorescence polarization measurements were

performed in a microplate reader (PHERAstar FS, BMG

Labtech) with an optical module equipped with polarizers and

using excitation and emission wavelengths of 546 and 563 nm,

respectively.

In order to confirm the oligomerization state, analytical

SEC with online multi-angle laser light scattering, absorbance

and refractive index (MALLS/UV/RI) measurements were

carried out on an Alliance 2695 HPLC system (Waters) using a

Silica Gel KW802.5 column (Shodex) equilibrated in 10 mM

HEPES, 150 mM NaCl pH 7.5. Detection was performed using

a triple-angle light-scattering detector (miniDAWN TREOS,

Wyatt Technology), a quasi-elastic light-scattering instrument

(DynaPro, Wyatt Technology) and a differential refractometer

(Optilab rEX, Wyatt Technology). Molecular-weight and

hydrodynamic radius determination was performed by the

ASTRA V software (Wyatt Technology) using a dn/dc value of

0.185 ml g�1. Proteins were loaded at a final concentration of

5 mg ml�1.

2.2. Protein crystallization, data collection and processing

Crystallization trials were performed by the hanging-

drop vapour-diffusion method at 293 K using a nanodrop-

dispensing robot (Mosquito, TTP Labtech). Optimal crystal-

lization conditions were obtained by mixing 100 nl protein

solution at 25 mg ml�1 with 100 nl reservoir solution (0.1 M

sodium acetate pH 5, 2 M ammonium sulfate). The crystals of

MERS-CoV NTD+ were flash-cooled in liquid nitrogen at

110 K. Glycerol was added to the mother liquor as a cryo-

protectant to a final concentration of 20%.

Diffraction data for the native enzyme were collected on

the ID30A-1 beamline at the European Synchrotron Radia-

tion Facility (ESRF), Grenoble, France using a Pilatus3 2M

detector at a wavelength of 0.965 A and a temperature of

100 K.

The data were integrated by XDS (Kabsch, 2010), indexed

in space group C121 (unit-cell parameters a = 208.648,

b = 67.021, c = 60.94, � = � = � = 90), scaled and truncated to a

resolution of 2.4 A assuming a minimum accepted signal-to-

noise ratio equal to 2. The data were subsequently used for

molecular replacement with Phaser for phasing and were

refined and validated by PHENIX (Adams et al., 2010). The

molecular-graphics software Coot (Emsley & Cowtan, 2004)

was used for model building, while Chimera (Pettersen et al.,

2004) was used to visualize and draw the models.

2.3. Small-angle X-ray scattering (SAXS) measurements anddata processing

All SAXS measurements were carried out on beamline

BM29 at the ESRF at a working energy of 12.5 keV, leading to

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194 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202

Figure 2Biophysical characterization of MERS-CoV NTD+. (a) Binding of RNAto MERS-CoV NTD+ assessed by fluorescence polarization. 30-Cy5-labelled RNA was incubated with various concentrations of NTD+. (b)MALLS/UV/refractometry/SEC analysis of NTD+. The right y axisrepresents the OD280 nm and the left y axis represents the molar mass ing mol�1 (Da). The absorption peak is shown as a noncontinuous line, withthe continuous trace indicating the molar mass. The value of themeasured mass at the volume corresponding to the top of the peak isreported.

a scattering vector s ranging from 0.025 to 5 nm�1. Data were

recorded using a Pilatus 1M detector at a sample-to-detector

distance of 2.43 m. The scattering vector s is defined as

s ¼4�

�sin �; ð1Þ

where � is the wavelength of the incident radiation in nano-

metres and � is half of the angle between the incident and the

scattered radiation.

A range of seven protein concentrations was measured

(0.21, 0.41, 0.77, 1.55, 3.06, 5.85 and 11.66 mg ml�1) in 10 mM

HEPES pH 7.5, 300 mM NaCl. All samples were centrifuged

for 15 min at 15 000g before the experiment in order to

minimize contributions from aggregated particles.

The measurements proceeded with 45 ml protein sample

injected into a 1.8 mm capillary with a flow to minimize

radiation damage; data were collected at 277 K.

Ten exposures of 1 s each were made for each protein

concentration and were combined to give the average scat-

tering curve for each measurement. A buffer reference was

performed before and after measurement for each protein

sample in the same conditions. The forward scattering inten-

sity was calibrated using BSA as a reference at 5 mg ml�1.

Data were processed with the ATSAS package (Petoukhov

& Svergun, 2007) according to the standard procedure. Ten

frames of 1 s per protein were averaged using PRIMUS

(Konarev et al., 2003). Frames affected by radiation damage

were excluded and the buffer background was subtracted from

the sample.

3. Results

3.1. Protein production and purification

NTD+ of the MERS-CoV N protein was partly designed

based on available coronavirus N-protein crystal structures

(Chen et al., 2007; Fan et al., 2005; Grossoehme et al., 2009;

Saikatendu et al., 2007). Sequence alignments coupled with

disorder predictions revealed that the N-terminal intrinsically

disordered region (IDR) starts at position 1 and ends at

position 36, whereas the structured RNA-binding domain

(RBD) encompasses amino acids 37–164 (Fig. 1a). To date, the

crystal structures of NTDs from different coronaviruses have

been obtained after the removal of the IDR. In this study, the

domain encompassing amino acid 1 to amino acid 164 was

used to produce NTD+ of the MERS-CoV N, thus including

the N-terminal IDR. The recombinant wild-type (wt) protein

was produced in E. coli and was purified for crystallization,

SAXS experiments and oligomerization studies. The quality of

the recombinant protein was assessed by a thermal shift assay

(TSA) and an RNA-binding assay using a nonspecific RNA

sequence. TSA experiments revealed a transition curve from a

folded to an unfolded state for MERS-CoV NTD+, suggesting

that the protein is structured. Moreover, the observed melting

temperature (Tm) is 313 K. For comparison, the Tm of the

MERS-CoV NTD+ is 8 K lower than that obtained for the

SARS-CoV NTD (Fang et al., 2009). An RNA-binding assay

using fluorescence polarization was performed as an addi-

tional quality assessment. The protein displays an affinity

constant in the hundred-nanomolar range for a nonspecific

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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 195

Table 1X-ray data-collection and refinement statistics.

Data collectionWavelength (A) 0.965Resolution range (A) 48.26–2.48 (2.486–2.480)Space group C121Unit-cell parameters (A, �) a = 208.648, b = 67.021, c = 60.94,

� = 90, � = 89.98, � = 90Total reflections 101472 (9834)Unique reflections 32509 (3088)Multiplicity 3.2 (3.2)Completeness (%) 98.04 (97.87)Mean I/�(I) 5.23 (1.90)Wilson B factor (A2) 55.81Rmerge 0.143 (0.6257)Rmeas 0.1708CC1/2 0.967 (0.831)CC* 0.991 (0.953)

RefinementRwork 0.2281 (0.4445)Rfree 0.2738 (0.4771)No. of non-H atoms

Total 4922Macromolecules 4839Ligands 32Water 51

No. of protein residues 621R.m.s.d., bonds (A) 0.005R.m.s.d., angles (�) 0.87Ramachandran favoured (%) 98.2Ramachandran outliers (%) 0Rotamer outliers (%) 0C� outliers (%) 0Clashscore 5.08Average B factor (protein) (A2) 64.70Overall MolProbity score 1.27

Figure 3Ribbon representation of the coronavirus NTDs. Superposition of theMERS-CoV NTD+ structure (blue) with those of the IBV (green) and theSARS-CoV (red) NTDs, showing the relatively good superposition of themain core of the protein and the high flexibility of the interconnectingrandom-coil loops.

RNA, with a Kd of 153 nM. The results are shown in Fig. 2(a).

Together, these results show that MERS-CoV NTD+ is well

folded and suitable for crystallization.

3.2. Structure determination by X-ray diffraction

Molecular replacement (MR) was carried out using the IBV

NTD (PDB entry 2btl; Fan et al., 2005). A unique solution was

found in space group C121 with five molecules per asymmetric

unit. Subsequent refinement converged with no particular

problems to an Rwork of 0.23 and an Rfree of 0.27. Details of

data collection and refinement are shown in Table 1. The

structure was deposited as PDB entry 4ud1.

The NTD+ domain is globular overall (fully �), presenting a

kidney-shaped surface organized as a main core featuring

three antiparallel �-strands forming a central �-sheet from

which a flexible �-hairpin structure

extrudes. These features are inter-

connected with flexible coil loops. The

flexibility of these coils is reflected by

elevated B factors (overall versus local)

and is also illustrated in Fig. 3, where the

MERS-CoV NTD+ structure (PDB

entry 4ud1, blue) is superimposed with

the IBV (PDB entry 2btl; Fan et al.,

2005) and SARS-CoV (PDB entry 1ssk;

Huang et al., 2004) NTD structures

(green and red, respectively). The first

30 residues of the N-terminal region of

MERS-CoV NTD+, corresponding to a

disordered region, could not be

resolved. This region was previously

scarcely resolved in all structures

obtained using X-ray diffraction tech-

niques, while it was resolved as far as

amino acid 24 in the 1ssk structure

measured in solution by NMR.

Fig. 4 shows the electrostatic surfaces

of several N-terminal RNA-binding

domains of NTD+ originating from

closely related viruses. Surfaces were

calculated by APBS and truncated from

�5 mV (red) to +5 mV (blue). Figs. 4(a)

and 4(b) show front and back views of

the protein, respectively. The common

feature of all structures is the positively

charged hairpin (oriented towards the

top side in the figure) and a hydro-

phobic core domain (Fig. 5b). By

contrast, the bottom side presents some

differences in the charge distribution,

especially for the proteins originating

from murine hepatitis virus and human

coronavirus OC43, which show a more

important negatively charged region

compared with the other structures (Ma

et al., 2010).

Analyses of the molecular arrange-

ment within the crystal show stacking of

Pro33 in front of the Trp43 residue

belonging to the nearby molecule. The

same stacking is also found within the

crystal packing of the IBV NTD struc-

ture as presented in Fig. 5. In these

figures hydrophobic residues (Val, Ile,

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196 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202

Figure 4Front (a) and back (b) views of electrostatic surfaces from several NTDs originating from closelyrelated viruses. The structure of the SARS-CoV NTD was measured in solution by NMR, while theother structures were all obtained by single-crystal X-ray diffraction. Surfaces were calculated byAPBS and truncated from �5 mV (red) to +5 mV (blue).

Leu, Phe, Trp, Cys, Ala, Tyr, His, Thr, Ser, Gly and Lys)

following the classification of Livingstone & Barton (1993) are

labelled in green and highlight the conserved hydrophobic

propensity of the core domain.

3.3. Oligomerization assays

Preparative SEC experiments that were carried out for

protein crystallization and RNA binding revealed that wt

NTD+ eluted predominantly as monomers. However, this

result is not sufficient to claim that the protein cannot behave

as a transient multimer. SEC-MALLS experiments indicated

that the observed molecular weight was 18 kDa, which is close

to the theoretical molecular weight (17.8 kDa), confirming the

SEC results (Fig. 2b). The hydrodynamic radius (Rh) was

equal to 1.85 � 0.45 nm. Although the MALLS data exclude

the possibility that multimers are formed after the protein

elutes from SEC, cross-linking experiments were also

performed with NTD+. The results revealed that even in the

presence of glutaraldehyde NTD+ has a very poor ability to

produce cross-linked multimers (data not shown). Altogether,

the oligomerization studies suggest that NTD+ of the MERS-

CoV nucleocapsid behaves as a monomer in solution and that,

as suggested by the small interface between NTD+ molecules

in the crystal, the interaction observed in the crystal does not

reflect an interaction in solution. In order to characterize the

behaviour of MERS-CoV NTD+ in solution as well as to

obtain structural information about the N-terminal part of

NTD+, SAXS experiments were also carried out.

3.4. Small-angle X-ray scattering (SAXS) measurements

3.4.1. Guinier analysis. Fig. 6 shows the Guinier analysis in

the range sRg < 1.3, where Rg is the radius of gyration

expressed in nanometres (Rg = 2 nm). The data corresponding

to concentrations (conc) from 1.55 to 11.66 mg ml�1 present

increasing nonlinearity with concentration. Data are repre-

sented as open circles, while red lines correspond to a linear

regression. The values of concentration in mg ml�1, Rg in

nanometres as well as the normalized forward scattering

intensity I(0)/conc are shown. Both Rg

and I(0)/conc increase with increasing

concentration, as is characteristic of an

interparticle interference term origi-

nating from attractive intermolecular

forces. However, for the lower concen-

tration data (0.2, 0.41 and 0.77 mg ml�1)

the above values stabilize at approxi-

mately Rg = 2 nm and I(0)/conc = 26.5.

Taking into account the calibration

SAXS profile of BSA, we estimate a

molecular weight (MW) of 25 kDa,

which is approximately 30% higher than

the expected protein MW of 17.8 kDa.

3.4.2. GNOM analysis. All concen-

trations were processed separately by

GNOM (Svergun, 1992) using the

PRIMUS interface. The results are

shown in Table 2. All parameters

increase together with the concentra-

tion, as observed by Guinier analysis in

the low-s region. Taking into account a

mean value of the Porod volume (V)

calculated for the three lower concen-

trations, hVi = 27.7 nm3, we estimate an

MW in the range 13.8 < MW < 18.4 kDa

(V/2 < MW < V/1.5), which is in accor-

dance with the MW of the protein

monomer (17.8 kDa).

Considering the three lower concen-

trations, the GNOM analysis gives a

radius of gyration Rg of 2 nm, which is

equal to that found by the Guinier

analysis. The ratio of Rg and Rh (Rg/Rh)

provides shape information about

proteins. Comparison with the hydro-

dynamic radius Rh of 1.85 nm measured

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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 197

Figure 5Representations of the interaction between NTD molecules in the crystal. (a) Ribbonrepresentation of the structures at the interface between NTD+ of MERS-CoV (left panel) andthe NTD of IBV (right panel) in which Pro33 of the nearby molecule (in purple) is stacked withTrp43 (highlighted in red). (b) Surface representation of the same structures as in (a), wherehydrophobic residues are labelled in green and the others in grey.

by MALLS gives an Rg/Rh ratio of 1.08. The usual Rg/Rh value

for a globular protein is�0.775, which means that Rg is smaller

than Rh. However, when molecules deviate from globular to

nonspherical or elongated structures then Rg/Rh tends to

values above 0.775 as Rg becomes larger than Rh. In the

present case the Rg/Rh value is probably owing to a deviation

from a globular structure owing to the additional N-terminal

region (amino acids 1–30) of the protein which protrudes out

of the main globular domain of the protein as discussed below.

3.4.3. EOM analysis (ensemble-optimization method). A

preliminary analysis with DAMMIF (Franke & Svergun, 2009)

of all four lower concentrations separately showed almost

identical particles that nicely fit the experimental data (data

not shown). The calculated particles feature a globular region

with a protuberance (Fig. 10b). The structure 4ud1 described

above nicely fits the globular part. In order to model the

supplementary protuberant part of the DAMMIF particle we

considered an additional 30-amino-acid N-terminal region and

used the EOM 2.0 program (Bernado et al., 2007; Tria et al.,

2015). Model PDB structures with an N-terminal region

containing the missing sequence were created. Two different

kinds of EOM strategy were used: (i) the N-terminal region

(amino acids 1–30) was modelled as a fully random region and

(ii) the N-terminal region was divided into three equally long

fully random parts spanning amino acids 1–30. The globular

part of the protein taken from the 4ud1 structure was kept

fixed in all cases. Both strategies gave the same results with

respect to the final model structures as well as the corre-

sponding Rg, size distributions, Rflex and R� statistics given by

EOM.

EOM created a pool of 10 000 possible particles with Rg and

size distributed randomly over the Gaussian-like distribution

profiles shown in red bold lines in Fig. 7(a). For each specific

concentration data set EOM chooses particle ensembles

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198 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202

Figure 7EOM analysis. (a) Rg and size distributions for each concentration (blacksticks) calculated by EOM. Pool distributions are shown as redcontinuous lines. (b) Superposition of several representative ensemblemodels calculated by EOM.

Figure 6Guinier analysis in the range sRg < 1.3 (Rg = 2 nm). Experimental data arerepresented by open circles, while red lines correspond to a linearregression. The values of concentration (conc), Rg in nanometres as wellas the normalized forward scattering intensity I(0)/conc are shown.Both Rg and I(0)/conc increase with increasing concentration, which ischaracteristic of an interparticle interference term originating fromattractive intermolecular forces.

Table 2Results of GNOM and AUTOPOROD analysis of all concentrationsseparately.

Concentration(mg ml�1) Rg (nm) I(0)/conc

Porod volume(nm3) Dmax (nm)

0.20 2.04 26.8 27.1 8.00.41 2.06 26.8 28.8 8.50.77 2.13 29.5 27.0 8.51.55 2.21 30.7 30.2 8.53.06 2.40 32.3 29.7 10.95.85 2.65 38.0 32.7 11.611.66 3.01 32.2 36.1 14.0

containing all possible conformations that fit the data. The Rg

and size distributions of these ensembles are shown as black

sticks. Fig. 7(b) depicts the typical ensemble models calcu-

lated. Models with high Rg (>2.2 nm) and large size (>8 nm)

correspond to molecules with a spatially extended N-terminal

region, while lower values of Rg and size correspond to

molecules with an N-terminal region folded closely to the

main core.

In order to enable a quantitative characterization of the size

or Rg distributions, EOM uses two metrics: Rflex and R� (Tria et

al., 2015). The Rflex metric is given as a percentage in the range

0–100%, with Rflex = 100% indicating maximum flexibility. The

R� metric indicates the variance of the ensemble distribution

with respect to the original pool and is defined as the ratio

�e/�p, where �e and �p are the standard deviations for the

distributions of the selected ensemble and the pool, respec-

tively. Values close to 1 describe a fully flexible system.

In Table 3 we report the Rflex and R� metrics of the

ensembles, as well as Rflex-pool showing the Rflex metric of

the pool, as a function of concentration between 0.2 and

3.06 mg ml�1. For each concentration the Rflex of the ensemble

ranges between 75 and 85% with an Rflex-pool of 90% and a R�in the range between 0.99 and 1.3. These values suggest the

presence of important flexibility as discussed by Tria et al.

(2015). However, visual inspection of the Rg and size distri-

butions in Fig. 7 shows the presence of a main ensemble

population centred around Rg = 2.2 � 0.2 nm as well as the

presence of a secondary ensemble population centred around

Rg = 1.7 � 0.2 nm which is clearly evident for the three lower

concentrations. The presence of these distinct ensembles

might indicate two preferential conformations of the

N-terminal region, which could be open or closed.

3.4.4. Analysis of a merged profile. Taking into account all

the above analysis, the data at the five lower concentrations

(0.2, 0.41, 0.77, 1.55 and 3.06 mg ml�1) were merged in order

to retrieve a single data file with good signal to noise. The

low-s region of the merged file is an average of the 0.2 and

0.4 mg ml�1 data, while the high-s region is an average of the

0.77, 1.55 and 3.06 mg ml�1 data.

A full analysis of the merged data with GNOM, DAMMIF

and EOM was undertaken. Details of this analysis are shown

in Table 4. A three-dimensional ab initio model was

constructed from the merged SAXS data using DAMMIF (s

range from 0.1 to 5 nm�1).

No predefined shape or symmetry was used. 20 different

models were calculated and subsequently averaged and

filtered by DAMAVER (Volkov & Svergun, 2003). Fig. 8

presents the resulting numerical fits of GNOM, DAMMIF and

EOM analysis. The GNOM fit curve is completely super-

imposed on the DAMMIF fit curve shown by the red contin-

uous line, while the EOM fit curve is shown by the blue

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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 199

Figure 8GNOM, DAMMIF and EOM fits. The GNOM fit is completelysuperimposed on the DAMMIF fit shown by the red continuous line,while the EOM fit is shown by the blue continuous line together with themerged data (open circles). The error signals, red dots for GNOM andDAMMIF and blue dots for EOM, are shown at the bottom of the figureand they have been almost superimposed for spatial economy.

Table 4SAXS data-collection and scattering-derived parameters.

The scattering and molecular-mass parameters are related to the merged dataprofile discussed in the text.

Data-collection parametersInstrument BM29, ESRFDetector Pilatus 1MBeam geometry (mm) 700 � 700Wavelength (A) 0.992q range (nm�1) 0.025–5Exposure time (s) 1Concentration range (mg ml�1) 0.2–11.66Temperature (�C) 4

Structural parametersI(0)/conc from P(r) (arbitrary units) 26.8I(0)/conc from Guinier (arbitrary units) 26.5Rg from P(r) (nm) 2.04Rg from Guinier (nm) 1.95Dmax (nm) 8Porod volume (nm3) 27.5

Molecular-mass determinationPartial specific volume† (nm3) 21.4Molecular mass from I(0)‡ (kDa) 24.1Molecular mass from sequence (kDa) 17.731

Software employedPrimary data reduction FIT2DData processing ATSASAb initio analysis DAMMIFValidation and averaging DAMAVERComputation of intensities CRYSOLThree-dimensional graphics CHIMERA

† The partial specific volume was taken to be (1.21 � MW) A3 per molecule (Harpaz etal., 1994). ‡ The molecular weight was calculated using calibrated data for BSA(66 kDa) at a concentration of 5 mg ml�1.

Table 3Rflex, Rflex-pool and R� metrics as a function of the concentration calculatedby EOM for the five lower concentrations separately.

Concentration (mg ml�1) Rflex (%) Rflex-pool (%) R�

0.20 76.8 91.1 0.990.41 75.1 90.4 1.300.77 78.8 90.5 1.201.55 84.7 90.1 0.953.06 85.2 89.4 1.32

continuous line along with the merged data (open circles). The

error signals, red dots for GNOM and DAMMIF and blue dots

for EOM, are shown at the bottom of the figure and have been

almost superimposed for spatial economy.

Fig. 9(a) shows the P(r) distribution of the merged data with

Dmax = 8 nm. Fig. 9(b) shows a Kratky plot of the merged data,

featuring an increase in the signal in the high-s region

(>2.5 nm�1), probably owing to the flexible N-terminal region.

In Fig. 10(a) we show the three ensemble structures

resulting from the EOM analysis of the merged data together

with the corresponding Rg and size distributions considered by

EOM (Fig. 10b). The contribution of each ensemble to the

measured SAXS profile is shown as a percentage under each

ensemble structure.

The corresponding metrics Rflex = 84% (Rflex-pool = 91%)

and R� = 1.12 correspond to a predominantly flexible

N-terminal region, while the size and Rg distributions shown

in Fig. 10(b) provide evidence for two distinct populations

(around Rg = 2.2 nm and Rg = 1.6 nm) as calculated previously

for each concentration separately. As a conclusion from the

EOM analysis, we can stress the presence of a flexible

N-terminal region together with the presence of two confor-

mations for the molecule: open and closed. In all cases the first

20 amino acids form a loop without being randomly unfolded.

The three ensemble model structures calculated by EOM fit

within the filtered particle calculated by DAMAVER as shown

in Fig. 10(c). The protuberant part of the particle mainly

corresponds to the open conformation with the N-terminal

region pointing out from the globular part.

4. Discussion and conclusion

MERS NTD+ encompassing amino acids 1–164 was crystal-

lized even though sequence prediction revealed the presence

of a disordered N-terminal region from amino acid 1 to amino

acid 37. Attempts to crystallize the NTD domain (from

positions 37 to 164) did not lead to suitable crystals from

commercial screens. It should be noted that during the writing

of this study a preliminary crystallographic analysis of the

NTD region encompassing amino acids 39–165 has been

published (Wang et al., 2015).

Our structural study therefore focused on the NTD+

domain, which could provide additional structural information

by combining crystal structure and SAXS analysis. Interest-

ingly, the asymmetric unit of the crystal was made up of five

NTD+ molecules organized with an interaction between Pro33

and Trp43 of the neighbouring molecule (Fig. 5). This inter-

action highlights the role of the intrinsically disordered region

(IDR) in the crystal organization and illustrates the need to

design several constructs for a given domain of interest in

order to improve the success rate at the level of protein

expression or crystallization (Bignon et al., 2013; Graslund et

al., 2008).

Although divergent in size and sequence, the NTDs for

which structures have been determined to date share a

common fold with a central �-sheet and a �-hairpin extension.

As expected, the three-dimensional structure of MERS-CoV

NTD+ displays the same structural organization. Its �-exten-

sion is positively charged (Fig. 4) and in several coronaviruses

it has been proposed to contribute to RNA binding through

electrostatic interactions with the phosphodiester groups of

the RNA (Fan et al., 2005). In addition to these interactions,

the conserved hydrophobic core domain could interact with

bases of the RNA through several aromatic residues (Huang

et al., 2004; McBride et al., 2014; Spencer & Hiscox, 2006).

Interestingly, in the crystal structure of MERS-CoV NTD+,

Trp43 located in the core domain was found to stack with a

proline (Pro33) of the adjacent NTD+, as in the crystal packing

of IBV NTD (Huang et al., 2004).

We therefore wanted to assess the oligomerization state

of MERS-CoV NTD+. SEC-MALLS analysis together with

SAXS data demonstrated that, at least under our experi-

mental conditions at pH 7.5, MERS NTD+ behaves mainly as a

monomer in solution. As previously reported, the oligomer-

ization of the MHV N protein is partly mediated by an

interaction between the NTD modules (Hurst et al., 2009).

Moreover, the NTD of the IBV N protein also has a weak

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200 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202

Figure 9Distance distribution and Kratky plot. (a) Distance distribution P(r) ofthe merged data with Dmax = 8 nm. (b) Kratky plot of the merged datashowing the characteristic increase of the signal in the high-s region(greater than 2.5 nm�1) owing to partial disorder of the molecule.

propensity for dimerization (Fan et al., 2005; Jayaram et al.,

2006). We can thus propose that, by itself, MERS-CoV NTD+

cannot promote MERS-CoV N-protein assembly, but could

contribute by a mechanism that still has to be unravelled to the

formation of the RNP through NTD–NTD interaction after

the CTD has already promoted the oligomerization.

The role of Trp43 in RNA binding has also been addressed.

MERS-CoV NTD+ binds to a nonspecific RNA in the

hundred-nanomolar range, and a W43A substitution did not

significantly alter the affinity constant (data not shown). RNA-

binding studies on MHV NTD revealed that aromatic residues

located in �3 and �5 are strong determinants for sequence-

specific RNA binding (Grossoehme et

al., 2009), whereas Trp43 precedes the

�1 strand. However, analysis of RNA

binding to the SARS-CoV NTD by

NMR showed that even for a poly-

adenine RNA the core region is still

involved in the binding (Huang et al.,

2004). It is therefore possible that

Trp43 could be involved in RNA

binding, but not as a main contributor,

and its single substitution does not allow

perturbation of the function. At this

stage, the role of Trp43, a conserved

residue in the betacoronaviruses, still

remains to be investigated.

SAXS experiments were undertaken

to characterize MERS-CoV NTD+ in

solution. The SAXS results showed

that the protein is monomeric and

composed of a globular part and a

flexible N-terminal region of 30 resi-

dues. This region is not completely

disordered but points out from the

globular part with two preferential

conformations: an open conformation

and a closed conformation. In all cases

the first 20 amino acids form a loop

without being randomly unfolded. Let

us recall that this N-terminus is involved

in RNA binding, working cooperatively

with the other RNA-binding sites of the

N protein (Chang et al., 2009). However,

it is possible that it plays other roles

since viral IDRs tend to be involved in

various interactions with host-cell

components other than RNA, such as

host membranes or proteins (Xue et al.,

2014).

Acknowledgements

This work was supported by the

SILVER Large Scale Collaborative

Project (grant agreement No. 260644) of

the European Union Seventh Frame-

work and the French Infrastructure for

Integrated Structural Biology (FRISBI)

ANR-10-INSB-05-01. The authors also

thank the ESRF for provision of

synchrotron beam time and the

MASSIF and BM29 staff for data

collection, as well as Stephanie Blangy

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Acta Cryst. (2016). D72, 192–202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid 201

Figure 10EOM analysis of a merged data set. (a) Three ensemble model structures resulting from EOManalysis of the merged data together with the corresponding percentage presence. (b) Rg and sizedistributions of the merged data. (c) Fit of the three structures within the filtered DAMAVERparticle.

and Silvia Spinelli for their contribution to the

experiments.

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202 Papageorgiou et al. � N-terminal part of the MERS-CoV nucleocapsid Acta Cryst. (2016). D72, 192–202